Feline panleukopenia vaccine

ABSTRACT

The attenuation of virulent strains of feline panleukopenia (FPL) viruses on ferret tissue and production of feline distemper (panleukopenia) vaccine on ferret tissue.

Simons et al.

July 1, 1975 FELINE PANLEUKOPENIA VACCINE Inventors: Robert W. Simons; William M.

Acree, both of White Hall, Ill.

[73] Assignee: Rohm and Haas Company,

Philadelphia, Pa.

[22] Filed: June 4, 1973 [21] Appl. N0.: 367,032

[52] US. Cl. 195/13; 424/89 [51] Int. Cl. C12k 7/00; Cl2k 9/00 [58] Field of Search 424/89; l95/l.3

[56] References Cited UNITED STATES PATENTS 3,293,l30 12/1966 Slator et al. 424/89 3,520,972 7/1970 Smith et al 424/89 3,562,387 2/197] Laubrman 424/89 OTHER PUBLICATIONS Andrews et al., Viruses of Vertebrates, 2nd Ed. (1967), pp. 370375 Panleukopenia of Cats (Syn onyms: Feline Infectious Enteritis ..Cat Distemper.- ..Enteritis of Mink) Pub. London, England, Bailliere Tindall and Lasseu.

Primary Examiner-Shep K. Rose Attorney, Agent, or FirmTerence P. Strobaugh [5 7] ABSTRACT The attenuation of virulent strains of feline panleukopenia (FPL) viruses on ferret tissue and production of feline distemper (panleukopenia) vaccine 0n ferret tissue.

6 Claims, N0 Drawings FELINE PANLEUKOPENIA VACCINE This invention relates to the attenuation of virulent strains of feline panleukopenia (FPL) viruses on Mustela tissue and production of feline distemper (panleukopenia) vaccine on Mustela tissue.

in the last few years, a number of techniques have been developed for the attenuation of virulent strains of panleukopenia viruses and subsequent production of a suitable vaccine. For the most part, these processes have suffered from the disadvantage that there are serious contamination problems with other, not easily detectable viruses. This is exaggerated since the conventional method of attenuation and subsequent vaccine production involves serially culturing a virulent strain of panleukopenia viruses on feline (usually kitten) tissue. It is not uncommon to carry out 20, 30 or more serial passages to obtain the necessary attenuation of the virulent virus to produce a seed virus. Since each serial passage requires the use Of fresh tissue, the process is extremely vulnerable to contamination with other viruses that may be present in the feline tissue. The same possibility of contamination is also present during the propagation stage or production stage of the vaccine. Recently, there have been indications that panleukopenia vaccine produced on feline tissue may also carry a leukemia virus which cannot be separated from the panleukopenia vaccine.

The objective of this invention is to provide a method of reducing the possibility of contamination of panleukopenia viruses during the attenuation stage and subsequent vaccine production stage. Other objects appear hereinafter.

We have now found that it is possible to carry out a substantial portion of the attenuation of panleukopenia virus on ferret (Mustela) tissue and thereafter produce the vaccine on ferret tissue. The replacement of part or all of the feline tissue has the advantage that it is possible to produce attenuated virus and vaccine without contamination from other viruses normally present in feline tissue. The ability to employ ferret tissue is surprising from the point of view that several workers in the field have indicated that it is not possible to culture panleukopenia virus on ferret tissue.

Briefly, applicants process of attenuating panleukopenia virus comprises serially l) acclimating the virus to in vitro growth on feline tissue followed by (2) further attenuation on Mustela tissue. It is essential that acclimated virus be brought into contact with a suspension of the ferret tissue cells. Failure to bring the virus into contact with suspended Mustela cells results in no virus reproduction. The resultant attenuated virus can then be used to seed Mustela tissue production batches. In the preferred method of operation, the titer of the Mustela attenuated seed virus is increased by inocculating feline tissue with the Mustela seed virus and harvesting to obtain a Mustela/feline seed virus for Mustela tissue production batches.

In somewhat greater detail, feline tissue cultures infected with a virulent strain of F PL virus are incubated for 2 to days at to C. depending upon the optimum growth rate of the cells. The virus laden fluids are harvested and used for subsequent passages Med. panleukopenia virus-free feline tissue cultures and ferret tissue cultures. For the purposes of all passages of the virus, the fresh tissue is obtained from disease-free animals. Typically, when an [CK-33 feline distemper virus strain obtained from D. L. Croghan at the 33rd passage level through cats is used as the source of the virulent strain, it is desirable to serially pass the virus through about 6 to 12 passages of feline tissue. For example, acclimation to in vitro growth may be attained by passing the virus through six feline lung tissue culture passages followed by two passages through feline embryo cells and two passages of feline kidney tissue. The last two feline kidney tissue passages are desirably employed in order to increase the virus titer. At this passage level, the virus produces a moderate fever and leukopenia indicative of feline distemper in susceptible kittens. However, the virus is sufficiently acclimated that it can be passed through ferret embryo tissue cultures and attenuated further. Typically, three passages on ferret tissue are sufficient to complete the attenuation and produce a seed virus. However, it is usually preferred to increase the virus titer at this stage by passing the virus through a feline kidney tissue culture passage. At this stage, the virus is attenuated, antigenic and immunogenic. This master seed virus can be employed to produce attenuated, antigenic and immunogenic feline panleukopenia vaccine using ferret tissue.

The growth medium in which the cells can be cultured include Parkers 199, Eagles, Earles, etc., either with or without the additional use of antibiotics such as penicillin, streptomycin, etc. The cells and virus are typically incubated at a temperature of about 30 to 45C., preferably 37C. i2C. The culture medium can be replaced at the end of about 24 to 240 hours from the time that the cells and virus are combined. Typically, the pH is maintained in the range of about 6.8 to 7.6, preferably about 7.2.

After the liquid containing virus is harvested from the culture of the first stage, it is further passaged in culture media containing tissue cells (cat and ferret during the virus attenuation stage and ferret tissues in the production stage). As in the first passage, the virus is contained in the growth medium of the same composition as previously specified, except for the feline tissue being replaced with Mustella tissues for a period of about 24 to 240 hours at a temperature of 30 to 45C., preferably 37C. fl".

Mustela or ferret tissue employed in this process can be from the embryo, lung or kidneys. Generally, ferret embryo tissue is employed during both the attenuation and production cycle.

While this invention is primarily directed to the attenuation of panleukopenia virus on Mustela tissue and propagation of panleukopenia vaccine on Mustela tissue, this invention can be employed to produce one or more batches of panleukopenia vaccine on Mustela tissue where the panleukopenia seed virus has been attenuated by any of the prior art methods, such as those described in U.S. Pat. Nos. 3,520,972; 3,293,130; etc., wherein feline tissue is used for attenuation. The disclosures of U.S. Pat. No. 3,520,972 of Smith, et al., and No. 3,293,130 of Slater, et al., are hereby incorporated by reference. However, it is essential that the Mustela cells must be in a suspended state (not in a monolayer) with the panleukopenia virus before there is cell growth. Failure to operate in this manner results in substantially no virus growth.

EXAMPLE 1 One ml. of ICK-33 feline distemper virus strain obtained from Dr. D. L. Croghaii at the 33fd passage level through cats was added to a tissue culture flask having 120 square centimeters of surface area together with approximately 2 X 10 feline lung cells and 25 ml. Basal Medium Eagles containing twice the normal amounts of vitamins and amino acids, 10% fetal bovine serum, 100 units penicillin and 100 mg. of streptomycin per ml. After 5 days at 37C., the virus fluids were harvested. The preceding is deemed to be passage 1. Passages 2-6 were carried out under identical conditions except that the virus fluid harvested from the preceding step was used in the succeeding step and the growth period was varied as set forth below in Table 1.

The virus fluids from passage 6 were purified by heating at 70C. for 30 minutes to inactivate viruses other than the feline panleukopenia virus.

The 7th and 8th serial passages were carried out in the same manner as passage 1 except that 2 X feline embryo cells were used in place of the feline lung cells and the 7th passage was maintained at 37C. for 5 days and the 8th passage was maintained at 37C. for 5 days. After the 7th passage, the virus was purified by filtering through a 50 millimicron membrane filter and heated for 3% hours at 75C. After the 8th serial passage, the virus was clarified by centrifugation, filtered through a 0.45 micron membrane filter and concentrated with Carbowax m. The concentrated virus was then ultracentrifuged using a cesium chloride gradient. The l 15th 1% ml. gradient out of 130 gradients was selected for further serial passage because of its relatively high potency and purity as compared to the other gradients.

For serial passages 9 and 10, 40,000 feline kidney cells were suspended in Minimum Essential Medium Eagles having non-essential amino acids and glutamine, 10% fetal bovine serum, 100 units penicillin and 100 mg. streptomycin per ml. together with the purified 1 15th gradient virus using a 1:1 ratio of virus particles (from the preceding passage) to cells. These were maintained at 37C. for 4-7 days prior to harvesting. The virus harvested from passage 10 was tested in kittens and found to produce mild panleukopenia.

At this stage the virus was sufficiently acclimated to in vitro culture that it could be grown on ferret tissue. Serial passages 1 1, l2 and 13 used a virus to cell ratio of 1:1. About 2 to 5 ml. of virus from the preceding passage and 30,000,000 cells of ferret embryo tissue in Minimum Essential Medium (Eagles), with nonessential amino acids and glutamine, 10% fetal bovine serum, 100 units of penicillin and 100 mg. of streptomycin/ml were suspended in roller culture bottles,

which were maintained at 37C. for 8 days and freezethawed by placing the cultures into a dry ice-acetone bath followed by thawing at 37C. thereby rupturing the cells and releasingcell-associated virus into the fluid.

The titer of the virus was then increased at passage No. 14 by using feline kidney cells and virus from passage 13. After maintaining the roller bottle culture at 37C. for 8 days, the culture was freeze-thawed in the manner described in the preceding paragraph. The resultant fluid virus was suitable for use as a master seed stock.

When kittens were vaccinated with the virus from the master seed stock, immunity was imparted to the kittens which prevented subsequent development of FPL at a later date, when challenged with a known virulent strain. I

EXAMPLE 2 This example illustrates the production of a panleukopenia vaccine using ferret tissue. 50,000,000 cells of ferret embryo tissue and 50,000,000 FAlD of virus, suspended in 2 liters of Minimum Essential Medium (Eagles), with non-essential amino acids and glutamine, l0% fetal bovine serum, units of penicillin and 100 mg. of streptomicinlml, were placed in a 9 liter roller bottle." After maintaining the culture at 37C. for 8 days, the vaccine was harvested using the freeze-thaw technique described in Example 1 yielding a vaccine capable of immunizing felines.

We claim:

1. In the process of producing attenuated feline panleukopenia vaccine, the improvement reducing the possibility of contamination with other feline viruses which comprises culturing an attenuated panleukopenia seed virus on ferret cell suspension tissue culture.

2. The process of claim 1, wherein said panelukopenia seed virus was attenuated by serial passages through feline tissue and ferret tissue, and virus from a serial passage through feline tissue was added to a cell suspension of ferret tissue.

3. The process of claim 2, wherein said panleukopenia seed virus was passed through a single passage through feline kidney tissue after the virus was attenuated through a cell suspension of ferret tissue.

4. In the process of producing an attenuated panleukopenia seed virus, the improvement which comprises providing a suspension of ferret cells and a virulent strain of panleukopenia virus and culturing on said suspended ferret cells said panleukopenia virus.

5. The process of claim 4, wherein said panleukopenia seed virus is attenuated on feline tissue to acclimate same and then serially passaged through a cell suspension of ferret tissue.

6. The process of claim 5, wherein the attenuated panleukopenia seed virus is passed further on feline tissue after serial passage through a cell suspension of ferret tissue.

UNITED STATES PATENT, OFFICE CERTIFICATE OF CORRECTION PATENT NO. 3, 9 7 DATED 1 July 1, 1975 INVENTOR(S) Robert W. Simone and William M. Acree It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1 line 63 Delete "Med. and in lieu thereof add Signed and Bealed this thirtieth Day of September1975 [SEAL] Affeif" RUTH C. MASON C. MARSHALL DANN 011w "mmlflwmr Parents and Trademarks 

1. IN THE PROCESS OF PRODUCING ATTENUATED FELINE PANTEUKOPENIA VACCINE, THE IMPROVEMENT REDUCING THE POSSIBILITY OF CONTAMINATION WITH OTHER FELINE VIRUSES WHICH COMPRISES CULTURING AN ATTENUATED PANLEUKOPENIA SEED VIRUS ON FERRET CELL SUSPENSION TISSUE CULTURE.
 2. The process of claim 1, wherein said panelukopenia seed virus was attenuated by serial passages through feline tissue and ferret tissue, and virus from a serial passage through feline tissue was added to a cell suspension of ferret tissue.
 3. The process of claim 2, wherein said panleukopenia seed virus was passed through a single passage through feline kidney tissue after the virus was attenuated through a cell suspension of ferret tissue.
 4. In the process of producing an attenuated panleukopenia seed virus, the improvement which comprises providing a suspension of ferret cells and a virulent strain of panleukopenia virus and culturing on said suspended ferret cells said panleukopenia virus.
 5. The process of claim 4, wherein said panleukopenia seed virus is attenuated on feline tissue to acclimate same and then serially passaged through a cell suspension of ferret tissue.
 6. The process of claim 5, wherein the attenuated panleukopenia seed virus is passed further on feline tissue after serial passage through a cell suspension of ferret tissue. 